The present invention relates to a novel and economical method for the preparation of chondroitin sulfate compounds. More particularly, the invention relates to a method for the preparation of chondroitin sulfate compounds including chondroitin sulfate A and chondroitin sulfate C having usefulness as an effective ingredient in eye lotions and therapeutic medicaments for neuralgia and arthralgia by using an inexpensive raw material available in large quantities.
Chondroitin sulfate is a generic name for a class of compounds known as a kind of typical glycosaminoglycans widely distributing in nature as a constituent of cartilage tissues and connective tissues of a great variety of animals. These compounds each have a polymeric structure consisting mainly of 40 to 100 times repetition of the disaccharide units expressed by the structural formula, the subscript n being the number of repetition of the disaccharide units: 
which can be classified into several types depending on the combinations and bonding positions of the substituents R1 to R4 including chondroitin sulfate A when R1 is SO3H and R2, R3 and R4 are each a hydrogen atom, i.e. chondroitin-4-sulfate, chondroitin sulfate C when R2 is SOH and R1, R3 and R4 are each a hydrogen atom, i.e. chondroitin-6 sulfate, chondroitin sulfate D when R2 and R3 are each SO3H and R1 and R4 are each a hydrogen atom, chondroitin sulfate E when R1 and R2 are each SO3H and R3 and R4 are each a hydrogen atom, chondroitin sulfate K when R1 and R4 are each SO3H and R2 and R3 are each a hydrogen atom and chondroitin sulfate B, i.e. dermatan sulfate, when most of the D-glucuronic acids are 5-epimerized into L-iduronic acid of which R1 is a sulfate group.
Among these chondroitin sulfate compounds, chondroitin sulfate A is prepared mainly from notochords of sturgeons or nasal cartilages of whales and chondroitin sulfate C is prepared mainly from cartilages of sharks as the respective starting raw materials. Availability of these raw materials, however, is limited and, in addition, the procedure for the preparation of the chondroitin sulfate compounds from these materials is relatively complicated not to be suitable for large-scale production of the products consequently with a high production cost.
The present invention accordingly has an object, in view of the above described problems in the conventional processes for the preparation of chondroitin sulfate compounds or, in particular, chondroitin sulfates A and C from raw materials of limited availability, to provide a novel and economical method suitable for large-scale preparation of these chondroitin sulfate compounds by a simple and convenient process from a raw material of good availability.
Thus, the method of the present invention for the preparation of chondroitin sulfate compounds or, in particular, chondroitin sulfate A and chondroitin sulfate C comprises the steps of:
(a) subjecting fish scales to an enzymatic solubilization treatment in an aqueous medium with a protease to give an aqueous solution of isolated chondroitin sulfate compounds containing polypeptides as a by-product;
(b) removing the by-product polypeptides from the aqueous solution obtained in step (a); and
(c) subjecting the aqueous solution after removal of the polypeptides to fractional precipitation of the chondroitin sulfate compounds.